Baculoviral Expression of ABCG2 Variants

The pFastBac1-BCRP vector and pENTR221-hBCRP entry vector for Gateway cloning were constructed as described previously (23 (link),24 (link)). The SNPs were incorporated into the ABCG2 gene in these vectors using the Q5 site-directed mutagenesis kit (New England Biolabs Inc., Ipswich, MA, USA) except for the 1582 G > A which was produced using the Q5 polymerase (New England Biolabs Inc.) and overlapping primers. DpnI (Thermo Fisher Scientific) was used to digest the template DNA. The presence of the SNPs was verified by sequencing the whole ABCG2 gene using the sequencing service from GATC Biotech (Constance, Germany). The ABCG2 WT and variant genes were transferred from the pENTR221-hBCRP entry vector to a modified Bac-to-Bac vector using Gateway cloning (Invitrogen). Recombinant baculoviruses for both Sf9 and HEK293 expression were generated according to the Bac-to-Bac protocol (Invitrogen Life Technologies, Carslbad, CA, USA). A vector containing the gene for enhanced yellow fluorescent protein (eYFP) was used as a control in the HEK293 expression system and an empty bacmid served as a control in the Sf9 system.